Download Isolation of infectious HIV-1

Isolation of infectious HIV-1
from culture-derived virus or
virus-containing sample
Special protocol
µMACS™ Streptavidin Kit
Index
1.2 Research applications
1.
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Description
1.1 Background
1.2 Research applications
1.3 HIV-1 capture strategy
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1.4 Reagent and instrument requirements
2.
Protocol for the isolation of HIV-1 virions
2.1 Magnetic labeling
2.2 Magnetic separation
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2.3 Elution option A for virion lysate
2.4 Elution option B for intact virions
3.
References
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1. Description
1.1 Background
The envelope of human immunodeficiency virus 1 (HIV-1) contains
not only virus-encoded proteins, but also host cell proteins.¹, ²
These host cell proteins are incorporated either actively or passively
when the virus buds from the cell membrane. Many of the cellular
proteins present in the HIV envelope retain their biological function,
suggesting that they could play a role in viral pathogenesis.¹ In
addition, the presence of certain host cell type-specific antigens
in the viral envelope serve as markers of the cellular origin of the
virus particle.³ MACS® Technology enables the rapid and efficient
magnetic isolation of infectious HIV-1 virions from culturederived HIV-1, human plasma or serum, and other bodily fluids, e.g.
cerebral spinal fluid or cervical lavage.
It has been determined that CD44, expressed on all leukocytes,
is the most effective host cell marker for the general labeling and
capture of HIV-1 from patient samples and culture-derived virus,
independent of the origin of the virus (lymphoid or myeloid cells).⁴
The µMACS™ VitalVirus HIV Isolation Kit enables the direct
isolation of HIV using CD44 MicroBeads. Virus originating from
distinct subpopulations of leukocytes can be isolated indirectly
using biotinylated antibodies directed against other cellular surface
antigens in combination with µMACS Streptavidin MicroBeads.⁴, ⁵
Accordingly, virus originating from lymphoid cells (T cell-derived
HIV) can be isolated with biotinylated CD26 antibodies; for virus
of myeloid origin (macrophage-derived HIV-1) biotinylated CD36
can be employed.
This protocol describes a magnetic-based method to isolate viable
and infectious HIV-1 for downstream studies utilizing biotinylated
CD26 or CD36 antibody and the µMACS Streptavidin Kit. For
isolation of HIV virions with CD44 marker, please refer to µMACS
VitalVirus HIV Isolation Kit user manual.
Order no. 130-074-101
Isolation of HIV-1 from patient samples for viral
compartmentalization studies from either T cells, by using a
biotinylated CD26 antibody, or from macrophages, by using a
biotinylated CD36 antibody.
Enrichment and detection of T cell- and/or macrophagederived HIV-1 from normal, diluted, and/or difficult samples,
e.g. cerebral spinal fluid, cervical lavage, breast milk. The
µMACS™ Streptavidin Kit can be used in conjunction with
commercially available kits for viral load analysis, see section
2.3, Elution option A.
Enrichment of live and infectious T cell- and/or macrophagederived HIV-1 for downstream studies, including neutralization
studies, and the development of primary isolates, see section
2.4, Elution option B.
Enrichment of T cell- and/or macrophage-derived infectious
HIV-1 in the presence of neutralizing antibodies or other
uncharacterized HIV serum inhibitors.
1.3 HIV-1 capture strategy
Isolation of T cell- and/or macrophage-derived HIV-1 from
human patient sample and culture-derived virus is performed by
positive selection using a biotinylated CD26 or CD36 antibody in
combination with the µMACS Streptavidin Kit. First, virions are
labeled with the antibody during a short incubation period, followed
by the labeling of the HIV-1 virions using the µMACS Streptavidin
MicroBeads. The magnetically labeled virions are enriched on a
µ Column in the magnetic field of a µMACS Separator.
1.4 Reagent and instrument requirements
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µMACS Streptavidin Kit (# 130-074-101)
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Phosphate-buffered saline (PBS) pH 7.2, supplemented with
0.5% bovine serum albumin (BSA) or 2% fetal bovine serum
(FBS)
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For enrichment of HIV of lymphoid origin: biotinylated CD26
antibody (clone 202.36 has been validated)
●
For enrichment of HIV of myeloid origin: biotinylated CD36
antibody (clone SMO has been validated)
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µMACS Separator (# 130-042-602)
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MACS® MultiStand (# 130-042-303)
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(Optional) 5 mL syringe plunger for elution of intact virions
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(Optional) 0.5% Igepal CA-630 (formerly NP-40) in PBS-lysis
buffer for viral antigen immunoassay³ for in-column viral lysis
and subsequent quantification, e.g. Innotest® HIV-1 Antigen
mAb, Innogenetics Inc.
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(Optional) Lysis buffer from commercial viral load assay kit
for in-column viral lysis and subsequent quantification, e.g.
NucliSens® EasyQ viral load assay, bioMerieux, Inc.
SP0014.02
Related products
●
µMACS™ VitalVirus HIV Isolation Kit (#130-092-805)
www.miltenyibiotec.com
Miltenyi Biotec GmbH
Friedrich-Ebert-Str. 68
51429 Bergisch Gladbach, Germany
Phone +49 2204 8306-0 Fax +49 2204 85197
Miltenyi Biotec Inc.
12740 Earhart Avenue, Auburn CA 95602, USA
Phone 800 FOR MACS, 530 8888871
Fax 530 8888925
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2. Protocol for isolation of HIV-1 virions
2.3.1 Elution for viral antigen immunoassay
2.1 Magnetic labeling
1.
Up to 1 mL virus-containing sample can be processed per isolation,
e.g. plasma, serum, cerebral spinal fluid, breast milk, or culture
supernatant. If fresh, non-frozen samples are used, virus isolation
should be started as soon as possible after collection to minimize
HIV-1 degradation.
Add 100 µL of lysis buffer, e.g. 0.5% Igepal CA-630 in PBS, to
the column. Incubate the column at room temperature for
5 minutes.
2.
Add an additional 150 µL of lysis buffer and collect all drops.
3.
Proceed to immunoassay.
1.
2.
2.3.2 Elution for viral load determination
For virus samples in saline solutions, e.g. vaginal lavage
samples: add non-specific blocking reagent, i.e. 0.5% BSA or
2% FBS.
1.
Briefly centrifuge sample, except breast milk samples, at
13,000×g for 30 seconds to remove particulate matter. Transfer
supernatant to a fresh tube, avoiding floating fragments.
Add 50 µL of lysis buffer, typically supplied in viral load assay
kit, to the column. Collect flow-through. Incubate the column
at room temperature for 5 minutes.
2.
Add an additional 150 µL of lysis buffer and collect all drops.
3.
Combine the eluates (total volume: 200 µL) and add 700 µL of
lysis buffer. Proceed to viral load determination assay.
▲ Note: Do not centrifuge breast milk samples as milk will separate.
3.
Add 1 µg of biotinylated CD26 or CD36 antibody per 200 µL viruscontaining sample, i.e. plasma, serum, or culture supernatant,
and incubate for 30 minutes at room temperature. For samples
of less than 200 µL volume, also add 1 µg of biotinylated
antibody.
▲ Note: Substitute CD26 or CD36 for isolation of compartmentalized HIV
derived from T cells or macrophages, respectively.
▲ Note: For isolation of virus from samples containing high concentrations
of natural biotin, e.g. breast milk, we recommend using 1.5 µg biotinylated
antibody per 500 µL sample. 5
4.
2.2 Magnetic separation
2.
2.4 Elution option B for intact virions
Perform elution of virus with column outside of magnetic separator
to obtain viable and infectious virus for downstream studies. HIV
virions remain infectious even with MicroBeads attached.
1.
Add 50 µL of µMACS™ Streptavidin MicroBeads per 200 µL of
sample and incubate for 10 minutes at room temperature. For
samples of less than 200 µL, adjust total volume to 200 µL with
PBS containing 0.5% BSA or 2% FBS before adding 50 µL of
µMACS Streptavidin MicroBeads.
▲ Note: For isolation of virus from samples containing high concentrations
of natural biotin, e.g. breast milk, we recommend using 75 µL of µMACS
Streptavidin MicroBeads or alternatively 100 µL of Anti-Biotin MicroBeads
(# 130-090-485) per 500 µL sample.⁵
1.
▲ Note: The NucliSens® EasyQ viral load assay requires 900 µL final volume
and supplies 900 µL aliquots of buffer.
Place a µ Column in the magnetic field of the µMACS Separator
that is mounted on the MACS® MultiStand.
Prepare the column by applying 100 µL of Equilibration Buffer
for protein applications, supplied with the µMACS Streptavidin
Kit, on top of the column.
3.
Rinse columns with 3×100 µL PBS containing 0.5% BSA
(or 2% FBS).
4.
Add the magnetically labeled sample from section 2.1.4 to the
column.
5.
Wash the µ Column with 4 × 200 µL of PBS containing 0.5% BSA
(or 2% FBS). Only add new buffer when the column reservoir
is empty.
Remove the column from the separator and place it on a
suitable collection tube, e.g. a 1.5 mL tube. Add 200–500 µL of
cell culture medium, or PBS, or other physiological buffer to
the column, and collect the eluate containing the magnetically
labeled virions.
▲ Note: To increase elution efficiency, apply pressure with 5 mL syringe plunger
after adding media/buffer.
3. References
1.
Tremblay, M. J. et al. (1998) The acquisition of host-encoded proteins by nascent
HIV-1. Immunol. Today 19: 346–351.
2.
Ott, D. E. (1997) Cellular proteins in HIV virions. Rev. Med. Virol. 7: 167–180.
3.
Lawn, S. D. et al. (2000) Cellular compartments of human immunodeficiency
virus type 1 replication in vivo: determination by presence of virion-associated
host proteins and impact of opportunistic infection. J. Virol. 74: 139–145.
4.
Lupo, L. D. and Butera S. T. (2004) Application of μMACS Streptavidin
MicroBeads for the analysis of HIV-1 directly from patient plasma. MACS&more
8(1): 16–19.
5.
Lupo, L. D. and Butera S. T. Unpublished observations.
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The products sold hereunder are warranted only to be free from defects in workmanship
and material at the time of delivery to the customer. Miltenyi Biotec GmbH
makes no warranty or representation, either expressed or implied, with respect to
the fitness of a product for a particular purpose. There are no warranties, expressed
or implied, which extend beyond the technical specifications of the products.
Miltenyi Biotec GmbH’s liability is limited to either replacement of the products or
refund of the purchase price. Miltenyi Biotec GmbH is not liable for any property
damage, personal injury or economic loss caused by the product.
SP0014.02
2.3 Elution option A for virion lysate
MACS is a registered trademark of Miltenyi Biotec GmbH.
Perform an in-column viral lysis for downstream quantification by
immunoassay or viral load determination. Buffer volumes should be
adapted to the downstream quantification assay. For expected virus
titres of > 10⁶ virions per mL, a p24 antigen immunoassay is adequate.
For expected titres of < 10⁶ virions per mL, a more sensitive viral load
assay is recommended. Examples are given below for the Innotest®
HIV-1 Antigen mAb, Innogenetics Inc. and for the NucliSens® EasyQ
viral load assay, bioMérieux, Inc.
µMACS is a trademark of Miltenyi Biotec GmbH.
Igepal is a registered trademark or trademark of various third parties.
Innotest is a registered trademark of Innogenetics Inc.
NucliSens is a registered trademark of bioMérieux, Inc.
© 2006 Miltenyi Biotec GmbH. Printed in Germany.
www.miltenyibiotec.com
Unless otherwise specifically indicated, all Miltenyi Biotec
products and services are for research use only and not for
diagnostic or therapeutic use.
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